BIOL 300 - Chapter 2

what are nucleases?

enzymes that degrade nucleic acid by breaking ester bonds in phosphodiester links; they’re the opposite of polymerases

what do endonucleases do?

cleave internal bonds

what do exonucleases do?

starts at end of strand and turns it into fragments

what is phosphatase?

enzyme that hydrolyzes ester bonds in nucleotide chains

what is the first type of restriction enzymes?

cleavage site is up to 1000bp away from recognition site

what is the second type of restriction enzymes?

recognition site and cleavage site are the same (inverse palindromic site); can use staggered or blunt cut

what are isochizomers?

enzymes that can produce either staggered or blunt cuts

what is the third type of restriction enzymes?

cleavage site is usually 20-30bp away from recognition site

what is a restriction map?

linear sequence of sites where restriction enzymes find their targets

what is recombinant DNA?

DNA molecule made up from 2+ sources

what is the purpose of cloning DNA?

used to sequence DNA and find ORF

what is a vector?

cloning vehicle - used to carry a desired piece of genetic material

what is an insert?

molecule to be cloned

what is PCR?

polymerase chain reaction - lab technique that makes copies of a segment of DNA from a small sample to detect and analyze discrepancies. thermal cycler that repeatedly denatures, anneals, extends, and repeats ~40 times

what are the unique features of vectors?

has an origin of replication, selectable marker, and multiple cloning sites

what is directional cloning?

uses two different restriction enzymes to leave different overhangs so vector and insert don’t self-circularize, and so orientation of how insert goes into vector is controlled.

what is ligating?

connecting the two pools of DNA fragments - needs 10:1 ratio of insert to vector

what is a liposome?

small sphere of artificial membranes that contain DNA or other biological materials

what is a probe?

allows for identification of a specific sequence of nucleic acid once it has a known sequence to target

what is in-situ hybridization?

procedure that allows scientists to look into cells and determine location of specific nucleic acid sequences

what are common tools used for studying DNA?

hybridization, cloning tools (PCR and vectors) and nucleases (with gel electrophoresis)

what is denaturation?

separation of two strands

what is Tm?

melting temperature - temperature where half of DNA helices are separated

what determines Tm?

DNA length, and G/C content

why does higher G/C content raise Tm?

G-C has 3 hydrogen bonds instead of two, which are harder to denature and take more heat/energy

what is hybridization?

the denaturation and renaturation of a strand

how does nucleic acid hybridization provide a way to recognize nucleic acid sequences in vivo and vitro?

through the principle of complementary base pairing. probes are designed to be complementary to the target sequence of interest.

what is FISH?

fluorescent in situ hybridization - uses fluorescent probe to allow it to spot target DNA more accurately

when are genes identified?

after the cloning process was completed

how does PCR amplify DNA genes?

repeated cycling through denaturation, annealing, and extension steps. Each cycle doubles the amount of the target DNA sequence, leading to exponential amplification

what is added to the 3’ end of DNA after DNA polymerase during PCR?

dNTPs - nucleotides with triphosphate (dATP, dGTP, dCTP, dTTP)

how is DNA visualized after PCR?

gel electrophoresis

how does gel electrophoresis separate DNA fragments?

by size - uses electric current to cause DNA to migrate to positive charge (shorter DNA moves faster, longer moves slower)

what is PCR commonly used for?

forensics (identification), gene research, identifying genetic disorders, sequencing genomes

what is Sanger sequencing?

DNA sequencing method that determines the order of nucleotide bases in a DNA molecule; uses ddNTPs to create DNA fragments of different lengths, which are then separated by size using electrophoresis. A laser then detects fluorescent ddNTPs, allowing software to convert these signals into the DNA sequence.

how can one study a gene that underwent PCR?

can manipulate the gene; isolate it to understand function or mutant genes, and make recombinant proteins

what are recombinant proteins?

proteins that are produced in a laboratory using genetic engineering techniques. They are created by inserting a gene of interest into a host organism, such as bacteria or yeast, which then produces the protein

what is recombinant DNA?

DNA from two different molecules that was cut with the same enzyme and pieced together

what can restriction nucleases be used for?

cleave DNA into defined fragments, and chew up bacteriophage DNA

what is the property of supercoiled DNA on agarose gel?

supercoiled DNA migrates faster than regular DNA on agarose gel

what are the common elements of cloning vectors?

origin of replication, centromere, marker (gene that confers a trait like resistance to a specific antibiotic), telomere, MCS (multiple cloning site / polylinker - a short DNA sequence containing several restriction enzyme recognition sites)

what are modified plasmids (cloning vectors) used for?

amplify DNA for seq, in vitro mods, identify promoter/enhancer elements, tag proteins to locate where they’re localized

why is cloning essential for making recombinant proteins?

expresses important proteins (purify large amounts of pure protein)